Oral VV116 versus placebo in patients with mild-to-moderate COVID-19 in China: a multicentre, double-blind, phase 3, randomised controlled study.

BACKGROUND: Spread of SARS-CoV-2 led to a global pandemic, and there remains unmet medical needs in the treatment of Omicron infections. VV116, an oral antiviral agent that has potent activity against SARS-CoV-2, was compared with a placebo in this phase 3 study to investigate its efficacy and safety in patients with mild-to-moderate COVID-19. METHODS: This multicentre, double-blind, phase 3, randomised controlled study enrolled adults in hospitals for infectious diseases and tertiary general hospitals in China. Eligible patients were randomly assigned in a 1:1 ratio using permuted block randomisation to receive oral VV116 (0·6 g every 12 h on day 1 and 0·3 g every 12 h on days 2-5) or oral placebo (on the same schedule as VV116) for 5 days. Randomisation stratification factors included SARS-CoV-2 vaccination status and the presence of high-risk factors for progression to severe COVID-19. Inclusion criteria were a positive SARS-CoV-2 test, an initial onset of COVID-19 symptoms 3 days or less before the first study dose, and a score of 2 or more for any target COVID-19-related symptoms in the 24 h before the first dose. Patients who had severe or critical COVID-19 or who had taken any antiviral drugs were excluded from the study. The primary endpoint was the time to clinical symptom resolution for 2 consecutive days. Efficacy analyses were performed on a modified intention-to-treat population, comprising all patients who received at least one dose of VV116 or placebo, tested positive for SARS-CoV-2 nucleic acid, and did not test positive for influenza virus before the first dose. Safety analyses were done on all participants who received at least one dose of VV116 or placebo. This study was registered with ClinicalTrials.gov, NCT05582629, and has been completed. FINDINGS: A total of 1369 patients were randomly assigned to treatment groups and 1347 received either VV116 (n=674) or placebo (n=673). At the interim analysis, VV116 was superior to placebo in reducing the time to sustained clinical symptom resolution among 1229 patients (hazard ratio [HR] 1·21, 95% CI 1·04-1·40; p=0·0023). At the final analysis, a substantial reduction in time to sustained clinical symptom resolution was observed for VV116 compared with placebo among 1296 patients (HR 1·17, 95% CI 1·04-1·33; p=0·0009), consistent with the interim analysis. The incidence of adverse events was similar between groups (242 [35·9%] of 674 patients vs 283 [42·1%] of 673 patients). INTERPRETATION: Among patients with mild-to-moderate COVID-19, VV116 significantly reduced the time to sustained clinical symptom resolution compared with placebo, with no observed safety concerns. FUNDING: Shanghai Vinnerna Biosciences, Shanghai Science and Technology Commission, and the National Key Research and Development Program of China. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

MicroRNA-4485 ameliorates severe influenza pneumonia via inhibition of the STAT3/PI3K/AKT signaling pathway.

The present study aimed to explore the potential roles and mechanism of microRNA-4485 (miR-4485) in severe influenza pneumonia. miR-4485 expression was detected in patients with severe H1N1 pneumonia using quantitative PCR. Furthermore, the effects of aberrantly expressed miR-4485 on H1N1-infected A549 cells were investigated using Cell Counting Kit-8, terminal deoxynucleotidyl transferase dUTP nick end labeling, western blotting and (ELISA) assays. Furthermore, the regulatory relationships between miR-4485 and the STAT3-mediated PI3K/AKT/mTOR signaling pathway were explored using a luciferase reporter and rescue assay. MiR-4485 expression was downregulated following H1N1 infection and in patients with H1N1 pneumonia. In addition, miR-4485 alleviated H1N1-induced A549 cell injury by promoting cell viability and the production of cytokines, as well as reducing apoptosis in A549 cells. Furthermore, STAT3 was revealed to be a target gene of miR-4485. Additionally, STAT3 silencing reversed the protective effects of miR-4485 knockdown on H1N1-induced cell injury via inhibition of the PI3K/AKT/mTOR signaling pathway. In conclusion, miR-4485 inhibited H1N1-induced severe pneumonia in A549 cells by targeting STAT3 via the PI3K/AKT/mTOR signaling pathway.

Clinical efficacy of nutrition support therapy combined with antibiotics in the patients of community-acquired pneumonia and its influence on serum pct and crp.

To analyze the clinical efficacy of nutrition support therapy combined with antibiotics in the treatment of patients with ICU severe community-acquired pneumonia and its effect on serum procalcitonin (PCT) and C reactive protein (CRP). A total of 90 patients with ICU severe community-acquired pneumonia treated in hospital from September 2016 to June 2017.The patients were randomly divided into A group and B group 45 cases in each group. Both groups were given antibiotic treatment of azithromycin plus cefuroxime sodium in which the A group received enteral nutrition support therapy while the B group parenteral nutritional support therapy. Levels of serum prealbumin (PA), albumin (ALB), transferrin (TF), procalcitonin (PCT) and C reactive protein (CRP) before and after treatment were compared. Before treatment there was found no significant difference in serum PA, ALB and TF levels (p>0.05) while after treatment, the serum levels of PA, ALB and TF in the A group were significantly higher than those in the B group (p<0.05). The effective rate of the A group was 88.9%, higher than that of the B group (p<0.05). In patients with ICU severe community-acquired pneumonia, the treatment of enteral nutrition support therapy combined with antibacterial drugs of azithromycin and cefuroxime sodium can effectively improve the indexes of PA, ALB and TF. The reduce levels of serum PCT and CRP with the good prognosis is important in popularization and application in clinical practices.

Fascin and CK4 as biomarkers for esophageal squamous cell carcinoma.

BACKGROUND: Several studies have suggested that fascin, cytokeratin 14 and cytokeratin 4 may have significant roles as biomarkers for the progression and survival of esophageal squamous cell carcinoma (ESCC). MATERIALS AND METHODS: This study performed immunohistochemistry in tissue microarrays, profiling premalignant lesions and invasive tumors. RESULTS: Fascin increased across the following states as follows: normal-appearing epithelium (26%) to dysplasia (46%) to ESCC (68%), while CK4 was undetectable in ESCC (0%) compared to normal-appearing epithelium (45%) or dysplasia (41%). CK14 was elevated and invariant in expression. In regression analyses, compared to normal-appearing epithelium, higher fascin expression was associated with a 36% increased risk of dysplasia (odds ratio=1.36) and a 56% increased risk of invasive ESCC (odds ratio=1.56). CONCLUSION: Expression of fascin is up-regulated in the transformation from normal-appearing epithelium, through dysplasia, into invasive carcinoma. Expression of CK4, CK14 and fascin did not correlate with patient survival. Fascin has a potential role as an early detection biomarker and CK4 as a tumor marker in ESCC.

Global gene expression profiling and validation in esophageal squamous cell carcinoma and its association with clinical phenotypes.

PURPOSE: Esophageal squamous cell carcinoma (ESCC) is an aggressive tumor with poor prognosis. Understanding molecular changes in ESCC will enable identification of molecular subtypes and provide potential targets for early detection and therapy. EXPERIMENTAL DESIGN: We followed up a previous array study with additional discovery and confirmatory studies in new ESCC cases by using alternative methods. We profiled global gene expression for discovery and confirmation, and validated selected dysregulated genes with additional RNA and protein studies. RESULTS: A total of 159 genes showed differences with extreme statistical significance (P < E-15) and 2-fold differences or more in magnitude (tumor/normal RNA expression ratio, N = 53 cases), including 116 upregulated and 43 downregulated genes. Of 41 genes dysregulated in our prior array study, all but one showed the same fold change directional pattern in new array studies, including 29 with 2-fold changes or more. Alternative RNA expression methods validated array results: more than two thirds of 51 new cases examined by real-time PCR (RT-PCR) showed 2-fold differences or more for all seven genes assessed. Immunohistochemical protein expression results in 275 cases which were concordant with RNA for five of six genes. CONCLUSION: We identified an expanded panel of genes dysregulated in ESCC and confirmed previously identified differentially expressed genes. Microarray-based gene expression results were confirmed by RT-PCR and protein expression studies. These dysregulated genes will facilitate molecular categorization of tumor subtypes and identification of their risk factors, and serve as potential targets for early detection, outcome prediction, and therapy.

[Studies on hereditary epidemiology of cardia cancer in Shanxi province.].

OBJECTIVE: Studies on cardia-cancer caused by hereditary factors. METHODS: Case-control method was adopted, with information including name, sex, date of birth, date of death of all the I, II, III relatives of the patients, diagnosis and the treatment collected. The hereditary probability of cardia cancer and the separation degree were calculated by Falconer and Li-Mentel-Gart. RESULTS: (1) Prevalence rates of cardia-cancer on relative I, relative II, relative III of cardia-cancer patients appeared to be 0.54%, 0.04%, and 0.05% respectively. Prevalence rates of upper-digestive-tract-cancer of relative I, relative II, relative III of cardia-cancer patients showed as: 2.50%, 0.36% and 0.13% respectively. Data showed that relative I > relative II > relative III and family cluster existed in both males and females. (2) Cardia-cancer hereditary probability of the relative I cardia-cancer probands was 11.71%, with males as 14.01% and females as 14.72%. The upper-digestive-tract-cancer hereditary probability of the relative I cardia-cancer probands was 13.87%, with males as 11.49% and females as 23.08%, both below 25%, indicating this was a low hereditary cancer. (3) The upper-digestive-tract-cancer separation of the blood compatriots of cardia-cancer patients was 0.0452, with males as 0.0441 and females as 0.0507, both below 0.25, indicating the nature of a multi-gene but not single-gene hereditary way. CONCLUSION: Hereditary factor is recognized as one of the high risk cardia cancer, but not the most risky factor causing the high morbidity of cardia cancer in Shanxi province.

[A comparative study on the risks of esophagus-cancer patients among factors as blood relatives, paternal line, matriarchal and different sex].

OBJECTIVE: In order to provide new clues on the cause of esophagus-cancer through seeking for information among the relatives of esophagus-cancer-patients at high-risk, contrast analysis was carried out to compare the ORs between esophagus-cancer cases and the relatives of the patients. METHODS: Case-control study was adopted on 720 cases and 720 controls who were kin relatives of the patients. RESULTS: (1) Risk of the relatives to the esophagus-cancer-patient group (1.34% - 2.24%) was obviously higher than the control group (0.78% - 1.21%) (P < 0.01). In 1(st) grade relatives, the risk of parent's to the esophagus-cancer patients (6.11%) was obviously higher than the control group (2.97%) (P < 0.01). (2) According to the cascade analysis to the cases of both paternal and matriarchal, lines, results showed that the risks of both the paternal line (0.87% - 1.01%) and the matriarchal line (0.50% - 0.79%) in the group of esophagus-cancer cases were all obviously higher than the lines in the control groups (0.53% - 0.65%) and (0.38% - 0.47%). Data also showed that the risk among the male relatives of paternal line (eg: grandfathers', father's, uncles' etc.) in the group of cases was 2.68% while the matriarchal (eg: grandmother's, mother's, aunts' etc.) was 1.91%. Both figures were obviously higher than that in the control group (1.50% and 0.92%, P < 0.01). CONCLUSION: The risk factor of esophagus cancer of the next generation seemed higher if the father and his brothers or mother and her sisters having had esophagus-cancers.

Biomarkers of apoptosis and survival in esophageal squamous cell carcinoma.

BACKGROUND: Cancer of the esophagus is a deadly malignancy, and development of biomarkers that predict survival is an urgent need. The apoptotic pathways have been hypothesized as important in progression of esophageal squamous cell carcinoma (ESCC). We investigated a panel of proteins that regulate apoptosis as candidate of biomarkers of prognosis in ESCC. METHODS: Tissue microarray (TMA) including 313 surgically-resected cases of ESCC specimens was built for immunohistochemical interrogation. We evaluated seven genes in the FasL-Fas apoptotic pathway - FasL, Fas, FAS-associated death domain protein (FADD), phosphorylated-FADD, and caspase 8 and 10, and the antiapoptotic protein bcl-2. We studied pathway integrity and relations to risk and clinical factors, and determined the prognostic significance of each marker. RESULTS: Five markers showed strong inter-marker correlations (r > or = 0.28, p < 0.001), including FasL, Fas, FADD, and caspases 8 and 10. FasL and FADD also showed modest correlations with one or more cancer risk factors, but none of the markers was significantly associated with either tumor stage or lymph node metastasis, the only two clinical factors that predicted survival in these ESCC cases. Multivariate-adjusted proportional hazard regression models showed no association between protein expression and risk of death for any of the seven markers examined. CONCLUSION: Individual biomarkers in the apoptosis pathway do not appear to predict survival of patients with ESCC.

Genomic characterization of esophageal squamous cell carcinoma from a high-risk population in China.

Genomic instability plays an important role in most human cancers. To characterize genomic instability in esophageal squamous cell carcinoma (ESCC), we examined loss of heterozygosity (LOH), copy number (CN) loss, CN gain, and gene expression using the Affymetrix GeneChip Human Mapping 500K (n = 30 cases) and Human U133A (n = 17 cases) arrays in ESCC cases from a high-risk region of China. We found that genomic instability measures varied widely among cases and separated them into two groups: a high-frequency instability group (two-thirds of all cases with one or more instability category of > or =10%) and a low-frequency instability group (one-third of cases with instability of <10%). Genomic instability also varied widely across chromosomal arms, with the highest frequency of LOH on 9p (33% of informative single nucleotide polymorphisms), CN loss on 3p (33%), and CN gain on 3q (48%). Twenty-two LOH regions were identified: four on 9p, seven on 9q, four on 13q, two on 17p, and five on 17q. Three CN loss regions-3p12.3, 4p15.1, and 9p21.3-were detected. Twelve CN gain regions were found, including six on 3q, one on 7q, four on 8q, and one on 11q. One of the most gene-rich of these CN gain regions was 11q13.1-13.4, where 26 genes also had RNA expression data available. CN gain was significantly correlated with increased RNA expression in over 80% of these genes. Our findings show the potential utility of combining CN analysis and gene expression data to identify genes involved in esophageal carcinogenesis.

[Testosterone induces different-featured prostate hyperplasia in castrated and uncastrated mice].

OBJECTIVE: To study the different features of hyperplasia in castrated and uncastrated mice after testosterone (T) treatment. METHODS: Forty-eight BALB/c mice were randomly divided into 6 groups of 8 in each: castrated (A), uncastrated (B) , castrated + low T (C), uncastrated + low T (D), castrated + high T (E), uncastrated + high T (F). Groups C and D were treated with testosterone solution at the dose of 12.5 mg/(kg d) and Groups E and F at 125 mg/(kg d) for 20 consecutive days, while Groups A and B received saline only. All the mice were sacrificed on the 21st day, their ventral and dorsal prostate glands weighed and their pathological features studied. RESULTS: Atrophic prostates were observed in Group A, but normal in Group B; prostatic hyperplasia was found in both Group C and D, but more obvious in the latter (P <0.05); and a slightly higher degree of hyperplasia was noted in Groups E and F than in C and D. There was an increase in serum T and vascular endothelial growth factor (VEGF) concentration and a decrease in serum estrogen (E2) concentration in the testosterone treated groups. CONCLUSION: Both castrated and uncastrated mice develop prostate hyperplasia after short-term testosterone treatment, although in different degrees and with different features, which may help further the studies on the association of castration and androgen with prostate diseases.

COX-2 (PTGS2) gene methylation in epithelial, subepithelial lymphocyte and stromal tissue compartments in a spectrum of esophageal squamous neoplasia.

BACKGROUND: Previous studies have shown important effects of stromal elements in carcinogenesis. To explore the tumor-stromal relationship in esophageal neoplasia, we examined methylation of COX-2 (PTGS2), a gene etiologically associated with the development of gastrointestinal cancers, in adjacent foci of epithelium, subepithelial lymphocytes and non-lymphocytic stromal cells found in sections of normal squamous epithelium, squamous dysplasia and invasive esophageal squamous cell carcinoma. METHODS: Adjacent foci of epithelium, subepithelial lymphocytic aggregates and non-lymphocytic stromal tissues were laser microdissected from six fully embedded, ethanol fixed, esophagectomy samples from Shanxi, China, a high-risk region for esophageal cancer. Promoter CpG site-specific hypermethylation status of COX-2 was determined using real-time methylation-specific PCR (qMS-PCR) based on Taqman Chemistry. The methylation status of a subset of samples was confirmed by pyrosequencing. RESULTS: Forty-nine microdissected foci were analyzed. COX-2 gene methylation was significantly more common in subepithelial lymphocytes (12/16 (75% of all foci)) than in epithelial foci (3/16 (19%)) or foci of non-lymphocytic stromal tissues (3/17 (18%)) (Fisher's exact p=0.05). Two of three epithelial samples and all three stromal samples that showed COX-2 methylation were adjacent to foci of methylated subepithelial lymphocytes. Pyrosequencing confirmed the methylation status in a subset of samples. CONCLUSIONS: In these esophageal cancer patients, COX-2 gene methylation was more common in subepithelial lymphocytes than in adjacent epithelial or stromal cells in both grades of dysplasia and in foci of invasive cancer. These findings raise the possibility that methylation of subepithelial lymphocytes may be important for tumorigenesis. Future studies of gene methylation should consider separate evaluation of epithelial and non-epithelial cell populations.

Overexpression of CDC25B and LAMC2 mRNA and protein in esophageal squamous cell carcinomas and premalignant lesions in subjects from a high-risk population in China.

Molecular events associated with the initiation and progression of esophageal squamous cell carcinoma (ESCC) remain poorly understood but likely hold the key to effective early detection approaches for this almost invariably fatal cancer. CDC25B and LAMC2 are two promising early detection candidates emerging from new molecular studies of ESCC. To further elucidate the role of these two genes in esophageal carcinogenesis, we did a series of studies to (a) confirm RNA overexpression, (b) establish the prevalence of protein overexpression, (c) relate protein overexpression to survival, and (d) explore their potential as early detection biomarkers. Results of these studies indicated that CDC25B mRNA was overexpressed (>/=2-fold overexpression in tumor compared with normal) in 64% of the 73 ESCC cases evaluated, whereas LAMC2 mRNA was overexpressed in 89% of cases. CDC25B protein expression was categorized as positive in 59% (144 of 243) of ESCC cases on a tumor tissue microarray, and nonnegative LAMC2 patterns of protein expression were observed in 82% (225 of 275) of cases. Multivariate-adjusted proportional hazard regression models showed no association between CDC25B protein expression score and risk of death [hazard ratio (HR) for each unit increase in expression score, 1.00; P = 0.90]; however, several of the LAMC2 protein expression patterns strongly predicted survival. Using the cytoplasmic pattern as the reference (the pattern with the lowest mortality), cases with a diffuse pattern had a 254% increased risk of death (HR, 3.52; P = 0.007), cases with no LAMC2 expression had a 169% increased risk of death (HR, 2.69; P = 0.009), and cases with a peripheral pattern had a 130% greater risk of death (HR, 2.30; P = 0.02). CDC25B protein expression scores in subjects with esophageal biopsies diagnosed as normal (n = 35), dysplastic (n = 23), or ESCC (n = 32) increased significantly with morphologic progression. For LAMC2, all normal and dysplastic patients had a continuous pattern of protein expression, whereas all ESCCs showed alternative, noncontinuous patterns. This series of studies showed that both CDC25B and LAMC2 overexpress RNA and protein in a significant majority of ESCC cases. The strong relation of LAMC2 pattern of protein expression to survival suggests a role in prognosis, whereas the association of CDC25B with morphologic progression indicates a potential role as an early detection marker.

[Expression and significance of GLUT-1 and DNA-PKcs in serous ovarian tumors].

BACKGROUND & OBJECTIVE: Cancer cell growth is supported by glucose metabolism of glucose transporter protein 1 (GLUT-1). In addition, the participation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in genome damage-repairing is important for oncogenesis prevention. This study was to evaluate the expression and biologic significance of GLUT-1 and DNA-PKcs in serous ovarian tumors. METHODS: The expression of GLUT-1 and DNA-PKcs in 20 specimens of normal ovarian tissues, 20 specimens of serous cystadenoma, 20 specimens of borderline serous cystadenoma, and 40 specimens of serous cystadenocarcinoma were detected by SP immunohistochemistry. The correlation of GLUT-1 and DNA-Pkcs expression to clinicopathologic characteristics of serous ovarian tumors was analyzed by Chi-square test. RESULTS: The positive rate of GLUT-1 was significantly higher in malignant and borderline serous ovarian carcinomas than in benign tumors and normal ovarian tissues (100% and 55% vs. 0% and 0%, P<0.01). The differences in the positive rate of DNA-PKcs were significant among normal ovarian tissues, benign, borderline, and malignant serous ovarian tumors (100%, 95%, 90%, and 60%, P<0.01). GLUT-1 expression was negatively correlated to DNA-PKcs expression (rs = -0.270, P<0.01). GLUT-1 expression in borderline and malignant serous ovarian tumors was related to intraperitoneal implants, ascites, lymph node metastasis, and FIGO stage, but DNA-PKcs was only related to FIGO stage and lymph node metastasis (P<0.05). CONCLUSION: The expression of GLUT-1 and the loss of DNA-PKcs may be closely related to the malignant transformation of serous ovarian tumors.

[Expression of GLUT-1, p63 and DNA-Pkcs in serous ovarian tumors and their significance].

OBJECTIVE: To investigate the expression of GLUT1, p63 and DNA-Pkcs in serous ovarian tumors and their significance. METHODS: GTUL1, p63 and DNA-Pkcs expression at protein level was detected by immunohistochemistry in patients with serous ovarian tumors. Chi-square analysis was used to assess if their expression is associated with clinicopathologic characteristics of the tumors. RESULTS: Cells in the normal ovarian tissues were not stained with GTUL1 and p63 antiserum, but DNA-Pkcs was positively stained. The intensity of GTUT1 and p63 expression was stronger in malignant ovarian serous tumors compared with other subtypes (P < 0.01). There were significant differences of DNA-PKcs among normal ovaries (100.0%), benign (95.0%), borderline (90.0%) and malignant (60.0%) serious ovarian neoplasms (P < 0.01). The level of GLUT-1 expression was correlated with FIGO staging, intraperitoneal implantation, ascites and lymph node metastasis (P < 0.05). p63 expression was associated with clinicopathologic characteristics except ascites (P < 0.05). DNA-PKcs was only correlated with FIGO staging and lymph node metastasis (P < 0.05). CONCLUSION: The results suggest that the abnormal expression of GTUT1, p63 and DNA-Pkcs may perhaps participate in serous ovarian tumor occurrence and development and may be considered as a marker reflecting tumor malignant behavior.

Genome-wide loss of heterozygosity and copy number alteration in esophageal squamous cell carcinoma using the Affymetrix GeneChip Mapping 10 K array.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a common malignancy worldwide. Comprehensive genomic characterization of ESCC will further our understanding of the carcinogenesis process in this disease. RESULTS: Genome-wide detection of chromosomal changes was performed using the Affymetrix GeneChip 10 K single nucleotide polymorphism (SNP) array, including loss of heterozygosity (LOH) and copy number alterations (CNA), for 26 pairs of matched germ-line and micro-dissected tumor DNA samples. LOH regions were identified by two methods--using Affymetrix's genotype call software and using Affymetrix's copy number alteration tool (CNAT) software--and both approaches yielded similar results. Non-random LOH regions were found on 10 chromosomal arms (in decreasing order of frequency: 17p, 9p, 9q, 13q, 17q, 4q, 4p, 3p, 15q, and 5q), including 20 novel LOH regions (10 kb to 4.26 Mb). Fifteen CNA-loss regions (200 kb to 4.3 Mb) and 36 CNA-gain regions (200 kb to 9.3 Mb) were also identified. CONCLUSION: These studies demonstrate that the Affymetrix 10 K SNP chip is a valid platform to integrate analyses of LOH and CNA. The comprehensive knowledge gained from this analysis will enable improved strategies to prevent, diagnose, and treat ESCC.

A multiplex tissue immunoblotting assay for proteomic profiling: a pilot study of the normal to tumor transition of esophageal squamous cell carcinoma.

Esophageal cancer remains a highly lethal malignancy for which the genetic and proteomic events are poorly understood. Studies have reported dysregulated proteins in esophageal carcinoma; however, the magnitude of these changes remains largely uncharacterized. Little is known about alterations early in the neoplastic pathway. Using multiplex tissue immunoblotting, we quantified the expression of seven proteins in esophageal carcinogenesis. Regions of normal, dysplasia, and invasive carcinoma of the squamous esophagus in six patients were characterized. Pan-cytokeratin (CK) was essentially unchanged across the transition (0.96 in dysplasia and 0.69 in tumor). Expression levels of annexin 1, CK-4, and CK-14 were all decreased in dysplasia and tumor compared with normal (reference, 1.00): annexin 1, 0.30 in dysplasia and 0.15 in tumor; CK-4, 0.20 in dysplasia and 0.16 in tumor; and CK-14, 0.54 in dysplasia and 0.40 in tumor. Expression of two proteins was increased in dysplasia and tumor versus normal: cyclooxygenase-2, 1.35 in dysplasia and 2.32 in tumor and p53, 1.29 in dysplasia and 2.37 in tumor. Secreted protein, acidic and rich in cysteine, which is expressed in the adjacent stroma, was 1.56-fold higher in stroma underlying dysplasia and 6.20-fold increased in dysplastic stroma surrounding invasive tumor. These findings suggest that changes in protein expression can be detected during the transition to dysplasia and may be useful biomarkers.

p16, MGMT, RARbeta2, CLDN3, CRBP and MT1G gene methylation in esophageal squamous cell carcinoma and its precursor lesions.

Esophageal squamous cell carcinoma (ESCC) is a common cancer with a very poor prognosis. New methods are needed to screen high-risk populations and identify curable tumors and precursor lesions early. Molecular markers may be useful in such screening efforts. This study was designed to determine the prevalence of p16, MGMT, RARbeta2, CLDN3, CRBP and MT1G gene methylation in patients with ESCC to evaluate the variation of gene methylation across a spectrum of preneoplastic lesions, and assess the feasibility of using gene methylation in a primary screening test utilizing frozen esophageal cells collected by balloon cytology samplers. Samples were obtained from high-risk subjects from north central China. These samples included 11 foci of histologically normal mucosa, 8 foci of low-grade squamous dysplasia, 7 foci of high-grade squamous dysplasia, and 13 foci of ESCC from 6 fully embedded resection specimens; endoscopic biopsies from 6 individuals with no histological evidence of disease; and frozen esophageal balloon samples from 12 asymptomatic subjects. Promoter CpG site-specific hypermethylation status was determined for each gene using real-time methylation-specific PCR (qMS-PCR) based on Taqman chemistry. Of the 6 ESCC patients, 5 showed methylation of at least one gene. For most genes, methylation occurred with increasing frequency during neoplastic progression, with the largest increase found between low- and high-grade dysplasia. There was considerable variation in methylation patterns among different foci of the same histological grade, even within individual patients, but 16/20 (80%) of high-grade dysplastic and cancer foci had >or= 2 methylated genes, while 17/19 (89%) of normal and low-grade dysplastic foci had <2 methylated genes. These genes were rarely methylated in histologically normal mucosa from patients with or without ESCC. Gene methylation was common and easily detectable in the frozen esophageal cells collected by balloon cytology samplers. Our data suggest that methylation of p16, MGMT, RARbeta2, CLDN3, CRBP, and MT1G is common in the esophageal mucosa of patients with ESCC in this high-risk population, and tends to increase in prevalence in foci with increasing histological severity of disease. Methylation data from panels of genes may be able to identify patients with high-grade lesions. Balloon cytology may be able to screen the length of the esophagus effectively for a subset of cells with abnormal methylation, and may be useful in a primary screening test for ESCC and its precursor lesions.

Accumulation of cytoplasmic beta-catenin correlates with reduced expression of E-cadherin, but not with phosphorylated Akt in esophageal squamous cell carcinoma: immunohistochemical study.

Accumulation of beta-catenin in cytoplasm occurs frequently during the pathogenesis of esophageal squamous cell carcinoma (ESCC). The mechanism leading to this alteration, however, is largely unknown. In the present study, immunohistochemistry was performed for beta-catenin, E-cadherin and Ser473 phosphorylated Akt (P-Akt) in 44 tissue samples of ESCC and corresponding normal esophageal epithelium. Exon 3 of the beta-catenin gene was analyzed by using single-strand conformation polymorphism and direct sequencing. In addition to the reduced expression of E-cadherin and membranous beta-catenin observed in 65.9% and 68% of ESCC tested, respectively, cytoplasmic accumulation of beta-catenin was also detected in 68% (30/44) cases. However, only two cases were found to have the same beta-catenin gene mutation. The data showed that cytoplasmic accumulation of beta-catenin was significantly associated with reduced expression of E-cadherin (P < 0.05) and that of membranous beta-catenin (P < 0.05). Furthermore, cytoplasmic beta-catenin was correlated significantly with lymph node metastasis (P < 0.05). In contrast, although strong staining of P-Akt occurred in 14 of 44 cases (32%), there was no significant correlation between the positive staining of P-Akt and cytoplasmic beta-catenin. Taken together these results suggest that the lost membranous beta-catenin might translocate to cytoplasm depending on reduced expression of E-cadherin, while Akt seems unlikely to play a role in this process.